Virus Surveys in Olive Orchards in Greece Identify Olive Virus T, a Novel Member of the Genus Tepovirus

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Article Virus Surveys in Olive Orchards in Greece Identify Olive Virus T, a Novel Member of the Genus Tepovirus

Evanthia Xylogianni 1, Paolo Margaria 2, Dennis Knierim 2, Kyriaki Sareli 1, Stephan Winter 2 and Elisavet K. Chatzivassiliou 1,*

1 Plant Pathology Laboratory, Department of Crop Science, School of Agricultural Production, Infrastructure and Environment, Agricultural University of Athens, Iera Odos 75, Votanikos, 11855 Athens, Greece; [email protected] (E.X.); [email protected] (K.S.) 2 Leibniz-Institute DSMZ-German Collection of Microorganisms and Cell Cultures GmbH, 38124 Braunschweig, Germany; [email protected] (P.M.); [email protected] (D.K.); [email protected] (S.W.) * Correspondence: [email protected]; Tel.: +30-2109-333-207

Abstract: Field surveys were conducted in Greek olive orchards from 2017 to 2020 to collect information on the sanitary status of the trees. Using a high-throughput sequencing approach, viral sequences were identified in total RNA extracts from several trees and assembled to reconstruct the complete genomes of two isolates of a new viral species of the genus Tepovirus (Betaflexiviridae), for which the name olive virus T (OlVT) is proposed. A reverse transcription–polymerase chain reaction assay was developed which detected OlVT in samples collected in olive growing regions in Central and Northern Greece, showing a virus prevalence of 4.4% in the olive trees screened. Sequences of amplified fragments from the movement–coat protein region of OlVT isolates varied from 75.64% to 99.35%. Three olive varieties (Koroneiki, Arbequina and Frantoio) were infected Citation: Xylogianni, E.; Margaria, P.; with OlVT via grafting to confirm a graft-transmissible agent, but virus infections remained latent. Knierim, D.; Sareli, K.; Winter, S.; In addition, cucumber mosaic virus, olive leaf yellowing-associated virus and cherry leaf roll virus Chatzivassiliou, E.K. Virus Surveys in Olive Orchards in Greece Identify were identified. Olive Virus T, a Novel Member of the Genus Tepovirus. Pathogens 2021, 10, Keywords: phylogenetics; HTS; ArMV; CLRV; CMV; OLYaV; OlVT; SLRSV; TMV; TNV 574. https://doi.org/10.3390/ pathogens10050574

Academic Editor: Akhtar Ali 1. Introduction Olives (Olea europaea L.) have been cultivated in the Mediterranean area since prehis- Received: 30 March 2021 toric times. Greek olive cultivation ranks third in the EU, after Spain and Italy [1], and the Accepted: 5 May 2021 olive agricultural and industrial sector is of immense importance for Greece, providing Published: 8 May 2021 economic, social and environmental services. Olive trees are mainly propagated using semi-hardwood cuttings. This procedure has contributed over the years not only to the dis- Publisher’s Note: MDPI stays neutral semination of the best olive materials, but also to the spread of pathogens, predominantly with regard to jurisdictional claims in viruses. Fifteen viruses have been reported to naturally infect olive trees [2], of which olive published maps and institutional affil- latent virus 1 (OLV-1), olive mild mosaic virus (OMMV) (Alphanecrovirus, Tombusviridae), iations. tobacco necrosis virus D (TNV-D) (Betanecrovirus, Tombusviridae), cucumber mosaic virus (CMV) (Cucumovirus, Bromoviridae), arabis mosaic virus (ArMV), cherry leaf roll virus (CLRV), olive latent ringspot virus (OLRSV) (Nepovirus, Secoviridae), tobacco mosaic virus (TMV) (Tobamovirus, Virgaviridae), olive latent virus 2 (OLV-2) (Oleavirus, Bromoviridae), Copyright: © 2021 by the authors. olive latent virus-3 (OLV-3) (Marafivirus, Tymoviridae), strawberry latent ringspot virus Licensee MDPI, Basel, Switzerland. (SLRSV) (Secoviridae) and olive vein yellowing associated virus (OVYaV) (Closteroviridae) This article is an open access article are officially recognized [3]. In addition, olive yellow mottling and decline associated distributed under the terms and virus (OYMDaV), olive semilatent virus (OSLV) and olive leaf yellowing associated virus conditions of the Creative Commons (OLYaV) have been proposed as distinct, but not yet approved, viral species [3]. In Greece, Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ graft-transmissible, virus-like diseases of olive trees have been reported [4]; however, up- 4.0/). to-date information on the occurrence of viruses in Greek olive orchards is scarce. Only

Pathogens 2021, 10, 574. https://doi.org/10.3390/pathogens10050574 https://www.mdpi.com/journal/pathogens Pathogens 2021, 10, x FOR PEER REVIEW 2 of 11

Pathogens 2021, 10, 574 2 of 11 however, up-to-date information on the occurrence of viruses in Greek olive orchards is scarce. Only recently, SLRSV, CLRV, OLYaV and ArMV were detected in a germplasm collection of the Greek Institute of Olive Tree, Subtropical Plants and Viticulture (IOSV) recently,in Chania SLRSV, (Crete) CLRV, [5]. In OLYaV earlier and studies ArMV reporting were detected from Greece, in a germplasm only OLV-3 collection was found of the to GreekinfectInstitute olive trees of Olive [6] with Tree, OLV-2 Subtropical infecting Plants Ricinus and Viticulture communis (IOSV)L. [7] and in Chania OMMV (Crete) infecting [5]. Inspinach earlier [8]. studies OLYaV reporting was identified from Greece, in onlyGreek OLV-3 olive wasvarieties found grown to infect in olivea Californian trees [6] withgermplasm OLV-2 infectingcollectionRicinus [9], although communis the originL. [7] andof the OMMV virus remains infecting unclear. spinach [8]. OLYaV was identifiedThis study in is Greek the first olive nationwide varieties grown survey in to a Californianinvestigate germplasmthe presence collection of viruses [9 ],in althoughGreek olive the orchards. origin of theVisual virus inspections remains unclear. during the survey did not reveal trees with con- spicuousThis studysymptoms is the indicating first nationwide viral infection survey and to investigate thus confirmed the presence the health of status viruses of inthe Greektrees. To olive support orchards. the survey, Visual a inspections global analysis during of olive the surveytranscriptomes did not was reveal performed trees with fol- conspicuouslowing a high-throughput symptoms indicating sequencing viral (HTS) infection approach and thus and confirmed subsequently the health a screening status ofby the trees. To support the survey, a global analysis of olive transcriptomes was performed RT–PCR. HTS permitted the discovery of a new viral species assigned to the genus Tepo- following a high-throughput sequencing (HTS) approach and subsequently a screening virus (Betaflexiviridae) for which the name olive virus T (OlVT) is proposed. RT–PCR tests by RT–PCR. HTS permitted the discovery of a new viral species assigned to the genus identified OlVT, CMV, CLRV and OLYaV infection in several olive trees. Tepovirus (Betaflexiviridae) for which the name olive virus T (OlVT) is proposed. RT–PCR tests identified OlVT, CMV, CLRV and OLYaV infection in several olive trees. 2. Results 2.2.1. Results Complete Genome Sequence Determination of a Novel Tepovirus 2.1. CompleteAnalysis Genome of the Sequence RNA-seq Determination data from ofth ae Novelpooled Tepovirus samples and BLAST alignments againstAnalysis a reference of the RNA-seqdatabase datarevealed from the the presence pooled samples of contigs and with BLAST similarity alignments to Tepovirus against amembers, reference databasein particular revealed to the the prunus presence virus of contigsT (PrVT) with isolate similarity Aze239 to [10]Tepovirus in poolmembers, MiV301 in(Table particular S1). No to theother prunus contigs virus with T (PrVT) homology isolate to Aze239viral sequences [10] in pool could MiV301 be identified (Table S1). by NoBLASTn other contigsor BLASTp with alignment. homology Screening to viral sequences of the RNAs could from be the identified samples by composing BLASTn orthe BLASTppool allowed alignment. us to trace Screening an infected of the plant, RNAs co fromrresponding the samples to sample composing GR168, the to pool its collection allowed usin toLamia, trace anFthiotida. infected Following plant, corresponding standard RT–PCR to sample and GR168, 5’-3´RACE to its reactions, collection a in complete Lamia, Fthiotida.genome of Following 6845 nt was standard assembled RT–PCR (isolate and GR168). 5’-3’ RACE The reactions,genome showed a complete an organization genome of 6845typical nt wasof members assembled of the (isolate genus GR168). Tepovirus The (Figure genome 1), showed containing an organizationthree overlapping typical open of membersreading frames of the genus(ORFs)Tepovirus that in direction(Figure1 ),5´ containingto 3´ encode three a replication-associated overlapping open reading protein frames(REP, position (ORFs) that 59–5395 in direction nt; 1778 5’ aa, to 3’ 205.4 encode kDa), a replication-associated a putative movement protein protein (REP, (MP, position position 59–53955307–6458 nt; nt; 1778 383 aa, aa, 205.4 42.6 kDa),kDa) and a putative a capsid movement protein (CP, protein position (MP, 6106–6771 position 5307–6458 nt; 221 aa, nt;25.2 383 kDa). aa, 42.6Two kDa) non-coding and a capsid regions protein (UTR) (CP, of 58 position nt and 6106–677174 nt in length nt; 221 are aa,present 25.2 kDa).at the Two5ánd non-coding 3´ ends of regions the genome, (UTR) respectively. of 58 nt and As 74 ntfor inother length members are present of the at Betaflexiviridae the 5’ and 3’ endsfamily, of thea polyA genome, tail respectively.follows the 3´ As UTR. for otherPrediction members of conserved of the Betaflexiviridae domains revealedfamily, the a polyApresence tail of follows expected the motifs 3’ UTR. in Prediction the viral pr ofoteins: conserved methyltransferase domains revealed (pfam01660, the presence aa 43– of334), expected 2OG-Fe(II) motifs oxygenase in the viral (pfam13532, proteins: methyltransferase aa 673–767), viral (pfam01660, RNA helicase aa 43–334),(pfamPF01443, 2OG- Fe(II)aa 959–1207), oxygenase RNA-dependent (pfam13532, aa 673–767),RNA polymerase viral RNA (pfam00978, helicase (pfamPF01443, aa 1355–1668) aa 959–1207),in the REP RNA-dependentprotein, the viral RNA movement polymerase protein (pfam00978, domain (pfam01107, aa 1355–1668) aa in 15–185) the REP in protein, the MP the protein, viral movementand the tricho-coat protein domain domain (pfam01107, (pfam05892, aa aa 15–185) 9–210) inin the the MP CP protein,protein. andAnalysis the tricho-coat of sample domainGR170 (Table (pfam05892, S1), collected aa 9–210) in in2019 the in CP the protein. same orchard Analysis (Lamia, of sample Fthiotida), GR170 allowed (Table S1), the collectedassembly in of 2019the complete in the same genome orchard of a second (Lamia, OlVT Fthiotida), isolate,allowed GR170. The the genome assembly is of6846 the nt completein length genomeand has ofthree a second ORFs with OlVT identical isolate, GR170.sizes to Thethe GR168 genome isolate, is 6846 with nt ina 5´ length UTR andof 58 hasnt and three a 3´ ORFs UTR with of 75 identical nt in length. sizes to the GR168 isolate, with a 5’ UTR of 58 nt and a 3’ UTR of 75 nt in length.

Figure 1. Schematic representation of the genomic organization of olive virus T isolate GR168. REP: replication-associated Figure 1. Schematic representation of the genomic organization of olive virus T isolate GR168. REP: replication-associated protein; MP: movement protein; CP: coat protein. Conserved motifs were predicted using Pfam (http://pfam.xfam. protein; MP: movement protein; CP: coat protein. Conserved motifs were predicted using Pfam org/search/sequence(http://pfam.xfam.org/search/sequence (accessed on 18 March (accessed 2021): on MeT, 18 Marc methyltransferase;h 2021): MeT, methyltransferase; AlkB, 2OG-FeII-Oxy2; AlkB, Hel,2OG-FeII-Oxy2; helicase; RdRp, Hel, RNA-dependenthelicase; RdRp, RNARNA-depe polymerase.ndent RNA polymerase.

Pairwise analysis at the whole genome level revealed 81.0% nt sequence identity between GR168 and GR170, and 74.5% and 74.8% nt identity, respectively, to the closest

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Pathogens 2021, 10, 574 3 of 11 Pairwise analysis at the whole genome level revealed 81.0% nt sequence identity between GR168 and GR170, and 74.5% and 74.8% nt identity, respectively, to the closest tepovirus, PrVT (KF700262). Alignment of the two OlVT isolates revealed 96.6% and tepovirus,84.4% identity PrVT in (KF700262). the 5´ and 3´ Alignment untranslated of the regions two OlVT (UTRs), isolates respectively. revealed At 96.6% the ORF and 84.4%nt/aa identitylevel, the in thehighest 5’ and values 3’ untranslated were observed regions for (UTRs),the CP, with respectively. 87.8% and At the91.4% ORF identity nt/aa level,at nt theand highest aa level, values respectively were observed (Table S2). for Phylogenetic the CP, with analysis 87.8% and of the 91.4% REP identityproteins atof ntaccepted and aa level,and putative respectively members (Table of S2). the Phylogeneticgenus Tepovirus analysis (Figure of 2A) the REPshowed proteins that the of acceptedolive isolates and putativecluster in members the clade of with the genusPrVT Tepovirusisolates, with(Figure the2 followingA) showed closest that the clade olive composed isolates clusterof zos- intera the virus clade T with (ZoVT) PrVT isolates. isolates, Sequence with the identity following at closestthe aa cladelevel composedwas 79.7–82.6% of zostera and 78.3– virus T83.5% (ZoVT) against isolates. PrVT Sequence accessions identity KF700263-KF7 at the aa level00262 was for 79.7–82.6% GR168 and and GR170 78.3–83.5% respectively. against PrVTThe CP-based accessions tree KF700263-KF700262 (Figure 2B) showed for the GR168 same and clustering GR170 with respectively. PrVT, while The the CP-based CP of treeZoVT (Figure was more2B) showed distant. the Amino same acid clustering identity withpercentages PrVT, while were the84.6–86.9% CP of ZoVT and 85.5–87.3% was more distant.aa against Amino PrVT acid accessions identity KF700262-KF70 percentages were0263, 84.6–86.9% for GR168 and and 85.5–87.3% GR170, respectively. aa against PrVT accessions KF700262-KF700263, for GR168 and GR170, respectively.

(A) (B)

FigureFigure 2. 2.Neighbor-joining Neighbor-joining phylogenetic trees trees constructed constructed on on the the deduced deduced aa aasequences sequences of the of replication-associated the replication-associated pro- proteinsteins (A (A) and) and capsid capsid proteins proteins (B ()B of) ofaccepted accepted and and putative putative members members of the of thegenus genus TepovirusTepovirus. The. The evolutionary evolutionary distances distances are represented as the number of amino acid substitutions per site (PrVT: prunus virus T; ZoVT: zostera virus T; ChVT: cherry are represented as the number of amino acid substitutions per site (PrVT: prunus virus T; ZoVT: zostera virus T; ChVT: virus T; PVT: potato virus T). cherry virus T; PVT: potato virus T). 2.2. Transmission of OlVT by Grafting 2.2. Transmission of OlVT by Grafting OlVT was transmitted by grafting scions from GR168 infected olive field tree to Koro- neiki,OlVT Arbequina was transmitted and Frantoio by graftingvarieties; scions however, from they GR168 remained infected symptomless. olive ﬁeld treeEight to Koroneiki,months after Arbequina grafting, and OlVT Frantoio was detected varieties; by however,RT–PCR they(Figure remained S1), and symptomless. the sequences Eight am- monthsplified from after the grafting, grafted OlVT plants was were detected identical by toRT–PCR those of the (Figure originally S1), and grafted the OlVT. sequences The ampliﬁedvirus was from not successfully the grafted plantstransmitted were identicalto the Arbosana, to those Mastoidis, of the originally Mavrolia grafted Messinias OlVT. Theand virus Coratina was notvarieties. successfully transmitted to the Arbosana, Mastoidis, Mavrolia Messinias and Coratina varieties. 2.3. Molecular Variability of OlVT 2.3. Molecular Variability of OlVT In total, six sequences from six trees were obtained by the Sanger sequencing of RT– In total, six sequences from six trees were obtained by the Sanger sequencing of RT– PCR products (GenBank accessions MΖ020972-MΖ020977), in addition to the sequence of PCR products (GenBank accessions MZ020972-MZ020977), in addition to the sequence of isolate GR170, which was afterwards subjected to HTS. Nucleotide sequence similarity in isolate GR170, which was afterwards subjected to HTS. Nucleotide sequence similarity the partial MP_CP region was in the range of 75.6–99.3%, with the highest diversity be- in the partial MP_CP region was in the range of 75.6–99.3%, with the highest diversity tween the isolates obtained in two successive years from the same tree in Fthiotida between the isolates obtained in two successive years from the same tree in Fthiotida (GR135OL and GR170) and the OlVT isolate from Korinthos (Peloponnese) (GR258OL) (GR135OL and GR170) and the OlVT isolate from Korinthos (Peloponnese) (GR258OL) (Figure 3). (Figure3).

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Figure 3. Maximum likelihood dendrogram depicting the phylogenetic relationships among the olive virus T’s (OlVT) partial movement protein and capsid protein sequences from the Greek isolates. Bootstrap values (%) analyzed by 1000 iterations are indicated on the corresponding branches.

2.4. Viruses in Greek Olive Orchards Visual inspections of olive trees in Greek orchards did not reveal conspicuous symptoms indicating viral infection. Viral testing by specific RT–PCR tests revealed viral infections in 13 out of the 158 samples (in total, 8.2% infection rate, Table S3). The novel OlVT was the most frequently detected virus in seven trees and five areas and was found in 4.4% of the tested trees. Besides Fthiotida (Lamia), OlVT was also detected in two distant fields in Argolida and one in each of Korinthia, Arkadia and Attica. Among the seven known olive viruses tested for, only three (CMV, CLRV, OLYaV) were detected: CMV was detected in five trees sampled in Agrinio, Attica, Argolida and Korinthia; OLYaV was detected in two trees, in Arkadia (Koutroufa) in the Peloponnese and Kilkis (Evropos) in Macedonia; and CLRV was detected in one tree in each of two areas (Megara and Paiania) in Attica (Figure4).

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FigureFigure 4. 4.Map Map of of Greece Greece showing showing samplingsampling areasareas (black(black dots)dots) withwith oliveolive orchards.orchards. Asterisks mark locations with OlVT-infected trees. locations with OlVT-infected trees.

2.5.2.5. Molecular Molecular Variability Variability of of Greek Greek Isolates Isolates of of Olive Olive Viruses Viruses 2.5.1.2.5.1. CMV CMV RdRp RdRp Gene Gene PhylogeneticPhylogenetic analysis analysis of of the the RdRp RdRp gene gene showed showed that that the the five five CMV CMV isolates isolates from from olive olive treestrees (GenBank (GenBank accessions accessions MW805377-MW805381) MW805377-MW805381) shared shared a 91.55–100% a 91.55–100% sequence sequence identity. iden- Onetity. CMVOne CMV isolate isolate (GR195OL (GR195OL from Paiania,from Paiania, Attica) Attica) was grouped was grouped in subgroup in subgroup IA of CMV; IA of theCMV; isolates the isolates from Korinthia from Korinthia and Argolida and Argolida in the Peloponnesein the Peloponnese (GR374OL, (GR374OL, GR375OL GR375OL and GR367OL)and GR367OL) were identicalwere identical and highly and similarhighly (99.16%)similar (99.16%) with that with of GR45OL that of fromGR45OL Agrinio, from andAgrinio, they wereand they all clustered were all in clustered CMV subgroup in CMV IB. subgroup The Greek IB. olive The isolatesGreek olive were isolates 85.55–89.1% were similar85.55–89.1% at the similar nt level, at withthe nt the level, only with three the olive only isolates three olive available isolates in available the GenBank, in the from Gen- AlbaniaBank, from (LR865402-LR865404). Albania (LR865402-LR865404). 2.5.2. OLYaV HSP70h Gene 2.5.2. OLYaV HSP70h Gene The amplification of the HSP70 homologue was successful in only one out of the The amplification of the HSP70 homologue was successful in only one out of the two two isolates of OLYaV (GR80OL) and resulted in a 339 nt sequence when excluding the isolates of OLYaV (GR80OL) and resulted in a 339 nt sequence when excluding the pri- primers (GenBank accession MW805382). This isolate showed higher sequence similarity mers (GenBank accession MW805382). This isolate showed higher sequence similarity (98.8%) with an isolate from the Ruget variety of French olive material kept in the USDA (98.8%) with an isolate from the Ruget variety of French olive material kept in the USDA germplasm collection in California (HQ286488). Our isolate showed only 75.89% sequence germplasm collection in California (HQ286488). Our isolate showed only 75.89% sequence similarity with the available Greek olive isolate (LR593885). similarity with the available Greek olive isolate (LR593885). 2.5.3. CLRV 3’ UTR 2.5.3. CLRV 3’ UTR Using the CLRV specific primers, we amplified an identical 341 bp long fragment from theUsing 3’ UTRthe CLRV region specific for the primers, two Greek we olive amplified isolates an (GR186OL identical 341 and bp GR252OL; long fragment Gen- Bankfromaccessions the 3’ UTR MW814913 region for andthe MW814914,two Greek olive respectively). isolates (GR186OL These sequences and GR252OL; showed onlyGen- 91.9%Bank similarityaccessions withMW814913 two other and identical MW814914, Greek resp oliveectively). isolates These already sequences publicly showed available only (MK936235,91.9% similarity MK936236). with two Higher other sequence identical similarity Greek olive (93.84%) isolates was already observed publicly with a Plantagoavailable major(MK936235,(KF779205) MK936236). and an Actinidia Higher sequence chinensis (KF779202)similarity (93.84%) isolate, both was fromobserved New with Zealand. a Plan- tago major (KF779205) and an Actinidia chinensis (KF779202) isolate, both from New Zea- land.

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3. Discussion Virus discovery studies in Greek olive orchards using HTS analysis enabled the assembly of the whole genome of a novel species of the genus Tepovirus [11] according to the species demarcation criteria in the Betaflexiviridae family [12]. In addition, graft transmission experiments unambiguously demonstrated its identity as a novel transmissible agent, for which the name olive virus T (OlVT) is proposed. To date, the genus Tepovirus includes potato virus T [11] and prunus virus T [3,10], while zostera virus T [13] and cherry virus T[14] have been recently suggested as new members. Total RNA was used as the input for library preparation, rather than dsRNA or sRNAs, which can be used in alternative HTS approaches [15] and have also been successfully employed for virus discovery in woody hosts [10,14,16,17]. The total RNA strategy was selected to take advantage of the potential for the broad discovery of both DNA and RNA viruses offered by this approach and because of the in-house bioinformatics pipeline optimized for long reads. The number of raw reads obtained per sample was in the range of ca. 0.8 × 106–2 × 106 and was sufficient to generate contigs that covered most of the genome of the two assembled OlVT isolates, whose complete sequence was determined by the complementary Sanger sequencing of amplification products. Analysis of the assembled OlVT genomic sequences clearly indicated the presence of two distinct isolates of the same viral species, with an identity percentage of 81% at nt level, similar to that observed for the PrVT isolates previously described [10]. A similar variability was observed on the partial MP_CP sequences of all obtained OlVT field isolates. The RT–PCR assay we developed allowed for the detection of OlVT in 4.4% of the tested trees (about half of those found to be infected by any virus), which were scattered in different areas in Central and Southern Greece. There are no known vectors for tepoviruses, and thus, the only known means of dissemination is infected vegetative propagation material. Nevertheless, similar to PVT [18], OlVT may be pollen- and seed-borne. In our graft transmission experiments, the important varieties Koroneiki, Arbequina and Frantoio became infected, but no symptoms were observed in the infected plants. The absence of symptoms is a common feature of most tepovirus infections and may facilitate OlVT spread via infected vegetative propagation material [2]. Other grafted varieties tested negative, which could be attributed to a lower viral load and/or sensitivity to OlVT; however, the number of plantlets tested was too low to safely conclude that. While graft transmission proved the infectivity of OlVT, additional studies are needed to record the sensitivity of further varieties and to assess any impacts of the viral infection. The virus survey results indicate that the infection rate in Greek olive orchards is rather low (8.2%) compared to that in other countries [19–24], where incidences can reach 74.6% (e.g., in Tunisia) [24]. However, a high virus incidence, e.g., 93.8% in California [9] is often recorded from surveys in germplasm collections, which may not reflect field infection rates. In Greece, among 40 young (4–5 years old) plants in a germplasm collection of the IOSV in Chania, 62.5% were infected [5]. Besides OlVT, only CMV, OLYaV and CLRV were detected in olive trees, but at low prevalence. CMV is endemic in many economically important crops in Greece [25]. CMV in olive trees has been reported from a few countries, always in latent infections; a higher infection rate (24.7%) has been reported from Egypt [23,26,27]. Although most Greek CMV isolates are clustered in subgroup IA, most of the olive isolates fell into subgroup IB, which includes isolates of East Asian origin [28] as well as some host-adapted CMV isolates from Greek vegetables [25]. There were no satellite RNAs associated with CMV isolates from olive trees. The olive-specific OLYaV, an unassigned species, possibly belonging to a new genus in the family Closteroviridae [29], was detected in two trees. Although this virus may be associated with a bright yellow discoloration in olive plants [2,26,30], changes in vegetative growth [31], or woody deformations [29], no such symptoms were observed. OLYaV appears to be one of the most widespread viruses in olives, with infection rates of up to 63% in Tunisia [2,24,26,32]. High OLYaV infection rates were also recorded in the germplasm repos- Pathogens 2021, 10, 574 7 of 11

itory of the USDA (National Clonal Germplasm Repository, Davis, California) where 93.8% of the tested varieties, including three Greek varieties, were found to be infected [9]. In Greece, 5% of the varieties kept in a germplasm collection of IOSV in Chania were infected with OLYaV [5]; however, with an isolate highly divergent from the one we collected. CLRV was also detected in Greek olive trees. The virus is known to cause severe disease in several of its numerous woody hosts [33]. CLRV is reported in olives and walnuts in Greece [5,34,35]. The virus is reported in latent infection in olives from several countries with infection rates reaching 15% in Syria [19,26,27] and is transmitted via olive seeds at a rate of 41% [36], which may also contribute to viral spread. Several “host-speciﬁc” clusters of CLRV isolates have been suggested [37]; however, the CLRV isolate we collected appears to diverge from the Greek olive sequences already available [5]. In this study, the nepoviruses ArMV and SLRSV were not found, although they were recently detected in trees from a Greek germplasm collection [5]. From our olive virus survey, we can report the identiﬁcation of a new virus species, OlVT, and the presence of a few known olive-infecting viruses in Greek orchards. None of the viral infections were associated with symptoms and the incidence was low. Contrasting the generally high virus incidence in “in situ” germplasm collections, the good health status of the Greek olive orchards can be stated. The fact that hardwood cuttings, the vegetative propagation materials, are the main means of viral spread, as well as the propagation of Greek autochthonous varieties by local nurseries and the, until recently, only very limited introduction of new materials from abroad may all be reasons for the low virus incidence. We found the new virus OlVT the most frequently, and this virus, which is not associated with symptoms, may have been present for quite some time in our country.

4. Materials and Methods 4.1. Plant Material In spring 2017, 42 samples were collected from olive trees from different areas in Greece (Table S4), for virus discovery by HTS. In the following years, from 2018 to 2020, surveys for olive viruses were conducted in early June or late September. A total of 158 trees were sampled, speciﬁcally in Northern Greece (Kilkis (5: the number of tested trees), Pella (3), Evros (2), Kavala (2), Kozani (2), Serres (1) Chalkidiki (1)), Central Greece (Attica (38), Aitoloakarnania (4), Arta (5), Fthiotida (32), Magnisia (5)), Southern Greece (the Peloponnese peninsula; Argolida (16), Arkadia (2), Korinthia (16), Messinia (5), Lakonia (1)), as well as on some islands (Naxos (1), Milos (1), Kimolos (2), Mykonos (1), Corfu (10), Lefkada (3)) (Figure4, Table S3). In order to assess the health status of the traditional Greek planting material, trees older than 20 years were considered for sampling. Because no conspicuous leaf symptoms could be observed, samples from up to 5 trees per ﬁeld were randomly and individually collected. From each tree, two 1- to 2-year-old twigs of 20–30 cm in length were collected from each of the four quadrants of the canopy. Trees to be used as negative controls in our tests were obtained from tissue culture (Vitroplant Italia srl Soc. Agricola, Italy) and kept in an insect-proof greenhouse.

4.2. HTS and Genome Assembly Total RNA was extracted from leaf tissues using an RNeasy Plant Mini Kit (QIAGEN, Germany), quality-checked by agarose gel electrophoresis and subsequently processed in the RNA-seq pipeline established at the DSMZ Plant Virus Department. The 42 samples collected in 2017 were processed in 11 pools (MiV300-310). An additional sample collected in 2019 that tested positive for OlVT in ﬁeld surveys by RT–PCR was sequenced as a single (Table S4). Ribosomal RNA was depleted using a RiboMinus Plant Kit (Invitrogen) according to manufacturer´s instructions. Following cDNA and second-strand synthesis using random octamers, an Illumina library was prepared following the NexteraXT Library Kit protocol and sequenced on a MiSeq system, following a 2X301 paired-reads strategy. Bioinformatic analyses for contig assembly, BLAST alignment, genome reconstruction and annotation were performed in Geneious Prime version 2021.0.1. A custom plant virus Pathogens 2021, 10, 574 8 of 11

database of RefSeq sequences downloaded from NCBI (generated on September 9th, 2020) was used as a reference for the local BLAST search. The complete genome sequences of the novel tepovirus were determined by the assembly of HTS contigs and Sanger-sequenced RT–PCR and 5’/3’ RACE products, obtained using primers designed on the partial genomic sequences in order to amplify PCR products spanning the gaps between the contigs or covering the terminal genomic regions (Table S5). The genome sequences of two OlVT isolates were submitted to GenBank under the accession number MW582811 (isolate GR168) and MW582812 (isolate GR170).

4.3. Detection of Olive Viruses by RT–PCR Trees were individually tested for the presence of ArMV, CLRV, CMV, OLYaV, OlVT, SLRSV, TMV and TNV-D using RT–PCR tests, with the primers reported in Table S6. Total RNA was extracted from 0.1 g of material from a mixture of cortical scrapings and leaves (including midrib) from each tree, which was powdered in liquid nitrogen prior to further processing using an RNeasy Mini Kit (QIAGEN, Germany). RNA was quantified in a NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific) and examined for integrity by agarose gels electrophoresis. The cDNA was synthetized from 1µg of total RNA using a RevertAid H Minus Reverse Transcriptase (Thermo Fisher Scientific) in the presence of 1.5 µg of random hexamers (Promega), in a 20µl reaction. Total RNAs extracted from freeze dried leaves infected with CMV, TMV, ArMV, TNV-D and SLRSV (DSMZ Plant Virus Collection), and OLYaV and CLRV RNA samples kindly provided by the Istituto per la Protezione Sostenibile delle Piante (Consiglio Nazionale delle Ricerche, Bari, Italy) were used as positive controls. For OlVT, a partial genome fragment cloned in a pDrive vector (QIAGEN, Germany) was used as positive control. RT–PCR was carried out in a total volume of 50 µL, containing 1µL of cDNA template, 0.2 µM of each primer (Table S6), 0.2 mM dNTPs, MgCl2 so as to have a final concentration optimized for each primer set (2 mM for consNecro, 2.5 mM for SLRSV and 3 mM for OlVT, OLYaV, CMV, CLRV and TMV) and 1.25 units of DreamTaq DNA Polymerase, in 1X Buffer (Thermo Fisher Scientific). NoRT reactions and cDNA synthesized from healthy plants served as controls. PCR conditions were as follows: initial denaturation at 95 ◦C for 3 min, followed by 35 cycles of denaturation at 95 ◦C for 30 s, annealing for 30 s at the appropriate temperature for each primer set and extension at 72 ◦C for 1 min, with a final elongation step of 72 ◦C for 5 min. PCR products were separated in 1.5% agarose gels, stained with ethidium bromide, purified with a Nucleospin Gel extraction kit (Macherey Nagel), and Sanger-sequenced in both directions by a commercial sequencing service provider (Applied Biosystems).

4.4. Sequence and Phylogenetic Analyses Pairwise similarities of OlVT with other tepoviruses were calculated according to the MUSCLE alignment on Sequence Demarcation Tool software (SDTv1.2) [38]. Phylogenetic trees on deduced amino acid sequences were generated in MEGA 7.0.25 [39] using the Neighbor-Joining method, with 500 bootstrap replicates, on a set of tepovirus sequences aligned using the online Clustal Omega tool (https://www.ebi.ac.uk/Tools/msa/clustalo/ (accessed on 18 March 2021). The molecular variability of the other viral sequences was investigated by retrieving related sequences from GenBank using BLASTn (http://www.ncbi.nlm.nih.gov (accessed on 25 March 2021). Subsequently, sequences were aligned using MUSCLE with a percentage Identity Matrix (PIM) calculated using the SIAS tool (http://imed.med.ucm.es/Tools/ sias.html (accessed on 25 March 2021)). MEGA version 7.0.26 [39] was used to infer evolutionary history using the Maximum Likelihood method, applying 1000 bootstrap iterations to validate the phylogenetic hypothesis of the Tamura-Nei model [40]. Pathogens 2021, 10, 574 9 of 11

4.5. Transmission of the Novel Tepovirus by Grafting Since OlVT is a novel virus for which no biological data exist, a conﬁrmation of the transmissibility of the agent was provided by grafting. Scions of infected trees were obtained from shoots collected in late February 2020, from an orchard tree infected with OlVT isolate GR168 (Lamia, Fthiotida). Scions were cleft-grafted onto two or three plantlets each of Koroneiki, Arbequina, Arbosana, Mastoidis, Mavrolia Messinias (from Kostelenos nurseries, Greece), Frantoio and Coratina (from tissue culture; Vitroplant Italia srl Soc. Agricola, Italy). All recipient trees used as rootstocks were previously tested and found to be virus-free. Grafted plants were subsequently kept in insect-proof greenhouses at ambient temperatures ranging from 10 ◦C to 30 ◦C. Samples from the newly developing vegetation of the rootstock were taken after 3 months (in May) and 8 months (in October) and tested by RT–PCR for the presence of OlVT.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/pathogens10050574/s1, Figure S1: Results of RT–PCR tests of olive plants grafted with olive virus T infected scions, Table S1: Overview of the HTS reads obtained from each sample library (raw, trimmed, normalized), viral contigs generated by de novo assembly, closest relative by BLAST analysis, and reads mapping the assembled final genome of the two olive virus T (OlVT) isolates (GR168 and GR170), Table S2: Percentage of identity of OlVT-GR170 and other (putative) tepovirus genomic sequences with OlVT-GR168, Table S3: Virus incidence in Greek olive orchards surveyed in 2018–2020 by RT–PCR, Table S4: Origin of the olive samples collected for virus discovery and composition of the pools used for HTS, Table S5: Primers designed on the partial olive virus T genomic sequences for determination of the whole genomic sequence by RT–PCR or RACE, Table S6: Primers used for the detection of olive viruses in Greek orchards by RT–PCR. Author Contributions: Conceptualization, E.K.C.; formal analysis, E.X., P.M., D.K., K.S., S.W. and E.K.C.; investigation, E.X., P.M., D.K. and K.S.; methodology, P.M., S.W. and E.K.C.; resources, E.K.C.; supervision, S.W. and E.K.C.; writing—original draft preparation, P.M., S.W. and E.K.C.; writing— review and editing, P.M., S.W. and E.K.C. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: All data generated or analyzed during this study are included in this published article (and its Supplementary Materials Files). Acknowledgments: The authors wish to thank M. Saponari (Istituto per la Protezione Sostenibile delle Piante, Consiglio Nazionale delle Ricerche, Bari, Italy) for the RNA positive controls for OLYaV and CLRV; G. Kostelenos nurseries for the olive plantlets of the Greek varieties used for the OlVT graftings; and Tryfon Ziatas and Panayotis Benardis for providing us with some olive samples. Conflicts of Interest: The authors declare no conflict of interest.

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